Insert alone gives colonies?

Chris Boyd cboyd at holyrood.ed.ac.uk
Mon Aug 21 09:56:20 EST 2000


No User <no.user at anon.xg.nu> wrote:
: Anybody ever seen something like this before? On a
: ligation of a long PCR fragment, much troubleshooting
: has shown that colonies come up on an insert alone +
: ligase control plate.  (Minipreps on colonies from the
: vector + insert plate are either vector without insert
: (rarely) or (mostly) some small garbage plasmid,
: smaller than the starting vector.  Also, a vector +
: insert without ligase control plate gives few or no
: colonies, suggesting that the colonies on the insert +
: ligase plate arise through ligation.)
: 
: The two possibilities I can think of are: 1)
: contamination of the insert with a rogue (linear)
: plasmid (somehow-- but everything is autoclaved,
: gel-isolated, not exposed to UV, treated carefully,
: etc.) or 2) somehow the insert itself is generating
: the plasmid (though you'd have to argue that somehow
: it's getting an amp resistance gene, an origin of
: replication, etc.-- seems pretty unlikely).  Any
: ideas?

It is most likely contamination of the PCR product with a linearised
plasmid -- perhaps the template itself -- that you co-isolated in the
gel slice.  Electrophoresis never gives perfect resolution, and
virtually any slice you take from a lane will contain at least a trace
representation of all the DNA you loaded (or in addition, if you didn't
clean out the tank carefully, a representation of DNAs loaded by your
colleagues in the recent past!).  That is why two or three rounds of gel
purification is often necessary.  Restriction analysis of the plasmid
from the insert + ligase colonies will be informative. 

Best wishes,
-- 
Chris Boyd                      | from, but \    Medical Genetics Section
Chris.Boyd at ed.ac.uk             | not for,  /    MMC, Edinburgh Uni.,
http://www.ed.ac.uk/~cboyd                       EH4 2XU, SCOTLAND
NEW! Edinburgh CF gene therapy jobs: http://www.jobs.ac.uk/jobfiles/LD218.html






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